Immunosuppressive myeloid cells in the tumor microenvironment (TME) inhibit T-cell-mediated immune response and promote tumor progression. Therapeutically targeting both tumor cells and myeloid cells such as myeloid-derived suppressor cells (MDSCs), is expected to promote antitumor immunity. Gemcitabine (Gem) can serve as a chemotherapeutic drug and a MDSC-depleting agent. Aberrant activation of STAT3 promotes tumor cell growth and orchestrates the immunosuppressive activity of tumor-associated myeloid cells. Here we describe a strategy to kill tumor cells as well as inhibit the expansion and suppressive function of myeloid cells through the systemic delivery of gemcitabine monophosphate (GMP) and STAT3 siRNA (siSTAT3). To enhance their in vivo delivery efficiency, we formulate GMP and siSTAT3 into a lipid-coated calcium phosphate (LCP) nanoparticle and a liposome-protamine-hyaluronic acid (LPH) nanoparticle, respectively. Compared to the control and monotherapy groups, combined GMP and siSTAT3 nanoparticles effectively induced tumor cell death, downregulated a wide range of pro-tumor signaling pathways and immunosuppressive mediators, eliminated MDSCs, enhanced T cell effector functions in tumors and lymphoid compartments, and led to superior therapeutic efficacy in a syngeneic mouse melanoma model. Additionally, these nanoparticles can serve as adjuvant treatment to improve the therapeutic response of anti-PD-1-based immune checkpoint blockade therapy. Thus, the combination of gemcitabine chemotherapy and STAT3 inhibition through nanotechnology could effectively kill tumor cells, alleviate the immunosuppressive TME, and enhance endogenous antitumor immunity.